The proposed research is for the development of assays utilizing immobilized bioluminescent enzymes from bacteria and fireflies, suitable for use in the clinical chemistry laboratory. Specifically with the bacterial system, if one covalently binds the NADH or NADPH specific FMN reductases along with luciferase, in principle any system generating NADH or NADPH can be assayed. The immobilized firefly luciferase can be used for assaying ATP in biological samples or for monitoring ATP formation in a coupled reaction. What is unique in the research proposed here is the use of immobilized luciferases which are stable and reusable for the detection of NADH, NADPH, and ATP. These immobilized systems offer the potential for a rapid and inexpensive method of assaying low concentrations of these biologically important compounds. Detection levels are several orders of magnitude lower than conventional spectroscopic methods.